DNA topoisomerases that were first discovered in 1971 (Wang, J. C., J Mol Biol., 55(3), 523-33, 1971) are essential enzymes in both prokaryotic and eukaryotic cells as they play key roles in genome topology maintenance. These nuclear enzymes catalyze DNA scission and religation reactions that either relax or supercoil DNA strands as well as remove knots and catenations, and in doing so produce different DNA topoisomers. They are believed to relieve torsional strain on DNA during replication and transcription processes. Because of their importance for cell viability, inhibitors of the topoisomerases (Topo) have been useful in treating cell proliferative conditions, in particular, human cancers because of the cytotoxicity they produce. The Topos have now been clearly identified as a validated molecular target for a variety of widely prescribed anticancer drugs (Pommier, Y., Cancer Therapeutics: Experimental and Clinical Agents, Teicher, B. A. ed., Humana Press, Totowan N.J., 153-174, 1997; Sausville, E., Anticancer Drug Development Workshop, DTP & NCI, N. Carolina, 2002). Collectively, the topoisomerase inhibitors comprise 6% of the total world market for cancer drugs in chemotherapy.
There are two general types of enzymes each of which has a different mechanism of DNA scission and different topological function, Topo I and II. The function of Topo I is to relax either positive or negative supercoiled DNA (Wang, J. C., Annu. Rev. Biochem., 65, 635-692, 1996) by cutting one strand of the duplex DNA, followed by strand passage of the intact strand, and finally religating the cut strand. Topo I does not require ATP hydrolysis for function. The strand-passage reaction requires that the enzyme be covalently attached to the DNA through a tyrosine residue (Ralph, R. K. et al., Topics in Molecular and Structural Biology: Molecular Aspects of Anti-cancer Drug-DNA Interactions, 1994). Topo I is not capable of more complex catalytic reactions such as catenation or decatenation because these processes require double strand cleavage.
Topo II acts on both strands of DNA by cutting; passage of a double-stranded DNA through the cleaved strands, followed by ligation and requires energy through ATP hydrolysis. It exists as two isozymes of 170 kD (Topo IIα) and 180 kD (Topo IIβ) (Drake, F. H. et al., J. Biol. Chem., 262(34), 16739-47, 1987; Drake, F. H. et al., Biochemistry, 28(20), 8154-60, 1989) and can relax supercoiled closed circular DNA plasmids in vitro (Osheroff, N. et al., J. Biol. Chem., 258(15), 9536-43, 1983). Topo IIα is highly expressed in proliferating cells and is located at the base of chromatin loops (Wood, E. R. et al., J Cell Biol., 111(6 Pt 2), 2839-50, 1990) implying a role in chromosome separation in mitosis whereas Topo IIβ is localized in the nucleolus implying a role in gene expression (Ura, K. et al., Nucleic Acids Res., 19(22), 6087-92, 1991) and its precise biological role is not known. Generally, many Topo IIα poisons also possess Topo IIβ poison activity (Austin C. A., Bioessays, 20(3), 215-26, 1998; Cornarotti, M. et al., Mol. Pharmacol., 50(6), 1463-71, 1996; Perri, D. et al., Biochem. Pharmacol., 56(4), 503-7 1998). Further, the significance of inhibitors specific for Topo IIβ such as XK469 (Gao, H. et al., Proc. Natl. Acad. Sci. USA., 96(21), 12168-73, 1999) has yet to be proven clinically.
Topo inhibitors can be divided into two classes by their mechanism of action: Topo poisons and reversible catalytic inhibitors. Topo catalytic inhibitors such as merbarone (Khelifa, T. et al., Mol Pharmacol., 55(3), 548-56, 1999) or ICRF-193 (Roca, J. et al., Proc. Natl. Acad. Sci. USA, 91(5), 1781-5, 1994) work by inhibiting the overall catalytic activity of the enzyme without introducing double strand DNA breaks, and work by inhibiting either the DNA binding, cleavage, or religation steps. Topo poisons are compounds that stabilize the covalent DNA-enzyme intermediates and therefore, turn it into a DNA damaging agent by introducing high levels of double-strand breaks which ultimately leads to triggering of apoptotic pathways (Kaufmann, S. H., Biochim. Biophys. Acta., 1400(1-3), 195-211, 1998). Topo poisons are known for both Topo I and II enzymes, and further, dual inhibitors of both Topo I and II are known to exist in each of the two general classes of inhibitors (Holden, J. A., Curr. Med. Chem. Anti-Canc. Agents, 1(1), 1-25, 2001).
Clinically relevant Topo II inhibitors act by trapping the enzyme in a covalent intermediate and can be divided into two classes. One class is comprised of DNA intercalators or minor-groove binders that impede the Topo religation step primarily by altering local DNA structure through directly binding DNA, and the second class consists of molecules that bind to the enzyme itself to prevent the DNA religation step.
Camptothecin is one of the first Topo I poisons to become a drug by binding to the Topo I-DNA complex reversibly (Leroy, D. et al., Biochemistry, 40(6), 1624-34, 2001). Second generation drugs to follow camptothecin are raltitrexed (AstraZeneca), irinotecan (Pharmacia), and topotecan (GlaxoSmithKline). Irinotecan has shown survival benefits in colorectal cancer as early as 1996, and is now a first-line use for colorectal cancer therapy and also has a major use in chronic lymphocytic leukemia. Topotecan has gained approval as a second-line treatment for ovarian and small cell lung cancer. These second generation drugs improve two problems with camptothecin, namely, the instability of the lactone ring in serum and low water solubility.
Anthracyclines, aminoanthracenes, podophyllotoxins, aminoacridines, ellipticines, and quinolones classes of molecules are known to act on Topo II. The most important drugs that target Topo II include the podophyllotoxins such as etoposide and the anthracyclines whose primary agent is doxorubicin, but amsacrine and mitoxantrone are marketed as well. Etoposide is front-line therapy for several malignancies including leukemias and lymphomas while the more widely used doxorubicin is front-line therapy in leukemias, lymphomas, and solid tumors including breast cancers. Second generation anthracyclines include daunorubicin (Gilead), and idarubicin (Pharmacia). The anthracyclines, in general, suffer from the major side effect of cardiotoxicity when administered chronically beyond certain accumulated doses. The second-generation compounds that follow doxorubicin are aimed at minimizing the cardiotoxic effects while maintaining anti-tumor effects. Second generation podophyllotoxins would include teniposide that is marketed as a drug.
Dual Topo I/II inhibitors which are furthest along in development would include: aclarubicin (NCI) which has completed phase II clinical studies, DACA (NCI) has phase I completed but halted, intoplicine (NCI) has completed phase I, TAS 103 (NCI/Taiho) is in phase I, BN80927 (Beaufour Ipsen) is in preclinical development, F11782 (Fabre) is also in preclinical, and XR11576 (Xenova/Millenium) which has completed phase I.
A significant problem that can arise in the clinic with most cancer treatments is the existence of multi-drug resistance (MDR). This can either be intrinsic when chemotherapy is started or acquired over time with treatment. The up-regulation of the MDR1 gene coding for P-glycoprotein (Pgp) is a major mechanism for acquired MDR (Endicott, J. A. et al., Annu. Rev. Biochem., 58, 137-71, 1989). Pgp is an ATP-dependent, membrane-bound drug efflux pump that has been extensively studied. Pgp has been shown to have broad substrate specificity and is capable of effluxing numerous xenobiotics and drugs including anthracyclines, Vinca alkaloids, epipodophyllotoxins, and taxanes from the cytoplasm of cells. Due to the key role of Pgp in MDR, antagonists of this pump are being discovered and tested for their use as an adjunct to anticancer drugs (Kaye, S. B. et al., J. Clin. Oncol., 16(2), 692-701, 1998; Sikic, B. I., Oncology (Hunting), 13(5A), 183-7, 1999) to increase penetration of antitumor drugs for MDR reversal. The net result of acquired MDR during chemotherapeutic treatment is after an initial remission in tumor growth, a diminished sensitivity to a broad range of drugs is seen presumably due to decreased intracellular concentrations, and tumors become refractory to treatment. Small-cell lung cancer is a good example of a malignancy that typically acquires MDR, and makes treatment very difficult.
Pgp is also thought to play a significant role in the establishment of the blood-brain barrier and is highly present in other tissues including liver, intestine, lung, kidney, and even bone marrow. Work performed on mice lacking their endogenous Pgp's (mdr1a/1b) provide compelling evidence that high levels of Pgp in a tissue can decrease the overall accumulation of drugs which are recognized by this efflux pump (Schinkel, A. H. et al., Proc. Natl. Acad. Sci. USA, 94(8), 4028-33, 1997).
Other drug resistance mechanisms specifically relating to Topo-directed drugs are: reduced Topo expression levels, Topo enzyme mutations, lengthened cell cycle time, diminished cellular accumulation, and altered DNA repair function (Chen, A. Y. et al., Annu Rev Pharmacol Toxicol., 34, 191-218, 1994). Also, inhibition of Topo I can induce increases in Topo II amounts, which can lead to resistance (Bonner, J. A. et al., Cancer Chemother Pharmacol., 39(1-2), 109-12, 1996). Therefore, there is still a need in the art for compounds that inhibit Topo I or Topo II or are dual inhibitors of both Topo I and II and is active against multi-drug resistant cancers. The present invention provides such new compounds, compositions and methods of treatment.